It doesn't give you a good information on what speeds to use and for how long. Do we just optimise it?I noticed that solution in the filter would be pulled down even by gravity and a 5 min spin at around 3000rpm would filter out most supernatant while it seems like the manual mentioned a time range between 30-45 min.

If i am right they hav given a fixed speed and varying time depending on the molecular weight cut off. i also just saw the manual from the same amicon 0.5ml centrifugal filter units, which we hav in our lab they do say the speed and time.

I have found that slower/longer spins are best, to prevent loss. I usually spin about 1K for 20-30 minute intervals until I achieve the desired final volume. sometimes it takes forever, but after all the other purification steps, I like to be conservative that close to the end

I have found that slower/longer spins are best, to prevent loss. I usually spin about 1K for 20-30 minute intervals until I achieve the desired final volume. sometimes it takes forever, but after all the other purification steps, I like to be conservative that close to the end

1K as in 1K rpm? or g?

Does the amount of time to take to purify a sample directly correlates to the abundance of the protein of interest in the sample?

How long it will take to concentrate is directly related to the size of your protein and the MWCO of the filter unit. I use the Amicon 0.5, 4, and 15ml concentrators and have never had a problem using them at the manufacturer's recommendation - just do shorter intervals of time then necessary so you're sure you don't over-concentrate. I've never reused amicon units but have with vivacells and rinsed them well and stored them in 20% ethanol in the fridge between uses.

The Amicon one's actually dry out due to gravity. So its hard to reuse them, hence the question. Maybe I can fill up both ontop (in the filter) and bottom in the tube to full capacity to avoid drying out?

Do you know if it's better to spin this centrifugal filter devices at low temperature (e.g. 4deg.C, double spinning time compared to at room temperature) so that the protein function can be maintained? Or it doesn't matter if it's used at room temperature?

I think it is protein dependent. Much of my work is with antibodies who behave fine at room temp so 4C isn't necessary but with some proteins crudely purified from lysates I've been more concerned and spun @ 4C and it was fine but it takes a long time. I wouldn't do it unless I had reason to believe it was necessary.

I think it is protein dependent. Much of my work is with antibodies who behave fine at room temp so 4C isn't necessary but with some proteins crudely purified from lysates I've been more concerned and spun @ 4C and it was fine but it takes a long time. I wouldn't do it unless I had reason to believe it was necessary.

Thanks Missle. My protein is crudely purified from yeast whole cell lysates. I guess I should spin it at 4degC then.